Affinity chromatography matrix

ABSTRACT

The present invention relates to a method of separating one or more immunoglobulin containing proteins from a liquid. The method includes first contacting the liquid with a separation matrix comprising ligands immobilised to a support; allowing the immunoglobulin containing proteins to adsorb to the matrix by interaction with the ligands; followed by an optional step of washing the adsorbed immunoglobulin containing proteins; and recovering said immunoglobulin containing proteins by contacting the matrix with an eluent which releases the proteins. The method improves upon previous separation methods in that each of the ligands comprises one or more of a protein A domain (E, D, A, B, C), or protein Z, or a functional variant thereof, with at least one of the monomers having a substitution of the C-terminal most proline residue after the third alpha-helix.

CROSS- REFERENCE TO RELATED APPLICATIONS

This application is a filing under 35 U.S.C. 371 of internationalapplication number PCT/SE2011/051432, filed Nov. 28, 2011, published onJun. 7, 2012 as WO 2012/074463, which claims priority to U.S.provisional patent application number 61/417,494 filed Nov. 29, 2010.

FIELD OF THE INVENTION

The present invention relates to the field of affinity chromatography,and more specifically to separation matrix containing ligand containingone or more of a protein A domain (E, D, A, B, C), or protein Z, with anamino acid substitution for the C-terminal most proline residue in atleast one of the monomers. The invention also relates to methods for theseparation of proteins of interest with aforementioned matrix, with theadvantage of increased capacity.

BACKGROUND OF THE INVENTION

Immunoglobulins represent the most prevalent biopharmaceutical productsin either manufacture or development worldwide. The high commercialdemand for and hence value of this particular therapeutic market haslead to the emphasis being placed on pharmaceutical companies tomaximise the productivity of their respective mAb manufacturingprocesses whilst controlling the associated costs.

Affinity chromatography is used in most cases, as one of the key stepsin the purification of these immunoglobulin molecules, such asmonoclonal or polyclonal antibodies. A particularly interesting class ofaffinity reagents is proteins capable of specific binding to invariableparts of an immunoglobulin molecule, such interaction being independenton the antigen-binding specificity of the antibody. Such reagents can bewidely used for affinity chromatography recovery of immunoglobulins fromdifferent samples such as but not limited to serum or plasmapreparations or cell culture derived feed stocks. An example of such aprotein is staphylococcal protein A, containing domains capable ofbinding to the Fc and Fab portions of IgG immunoglobulins from differentspecies.

Staphylococcal protein A (SpA) based reagents have due to their highaffinity and selectivity found a widespread use in the field ofbiotechnology, e.g. in affinity chromatography for capture andpurification of antibodies as well as for detection. At present,SpA-based affinity medium probably is the most widely used affinitymedium for isolation of monoclonal antibodies and their fragments fromdifferent samples including industrial feed stocks from cell cultures.Accordingly, various matrices comprising protein A-ligands arecommercially available, for example, in the form of native protein A(e.g. Protein A SEPHAROSE™, GE Healthcare, Uppsala, Sweden) and alsocomprised of recombinant protein A (e.g. rProtein A SEPHAROSE™, GEHealthcare). More specifically, the genetic manipulation performed inthe commercial recombinant protein A product is aimed at facilitatingthe attachment thereof to a support.

These applications, like other affinity chromatography applications,require comprehensive attention to definite removal of contaminants.Such contaminants can for example be non-eluted molecules adsorbed tothe stationary phase or matrix in a chromatographic procedure, such asnon-desired biomolecules or microorganisms, including for exampleproteins, carbohydrates, lipids, bacteria and viruses. The removal ofsuch contaminants from the matrix is usually performed after a firstelution of the desired product in order to regenerate the matrix beforesubsequent use. Such removal usually involves a procedure known ascleaning-in-place (CIP), wherein agents capable of eluting contaminantsfrom the stationary phase are used. One such class of agents often usedis alkaline solutions that are passed over said stationary phase. Atpresent the most extensively used cleaning and sanitising agent is NaOH,and the concentration thereof can range from 0.1 up to e.g. 1 M,depending on the degree and nature of contamination. This strategy isassociated with exposing the matrix for pH-values above 13. For manyaffinity chromatography matrices containing proteinaceous affinityligands such alkaline environment is a very harsh condition andconsequently results in decreased capacities owing to instability of theligand to the high pH involved.

An extensive research has therefore been focussed on the development ofengineered protein ligands that exhibit an improved capacity towithstand alkaline pH-values. For example, Gülich et al. (SusanneGülich, Martin Linhult, Per-Åke Nygren, Mathias Uhlén, Sophia Hober,Journal of Biotechnology 80 (2000), 169-178) suggested proteinengineering to improve the stability properties of a Streptococcalalbumin-binding domain (ABD) in alkaline environments. Gülich et al.created a mutant of ABD, wherein all the four aspargine residues havebeen replaced by leucine (one residue), asparte (two residues) andlysine (one residue). Further, Gülich et al. report that their mutantexhibits a target protein binding behaviour similar to that of thenative protein, and that affinity columns containing the engineeredligand show higher binding capacities after repeated exposure toalkaline conditions than columns prepared using the parentalnon-engineered ligand. Thus, it is concluded therein that all fourasparagine residues can be replaced without any significant effect onstructure and function.

Recent work shows that changes can also be made to protein A (SpA) toeffect similar properties. US patent application publication US2005/0143566 discloses that when at least one asparagine residue ismutated to an amino acid other than glutamine or aspartic acid, themutation confers an increased chemical stability at pH-values of up toabout 13-14 compared to the parental SpA, such as the B-domain of SpA,or Protein Z, a synthetic construct derived from the B-domain of SpA(U.S. Pat. No. 5,143,844). The authors show that when these mutatedproteins are used as affinity ligands, the separation media as expectedcan better withstand cleaning procedures using alkaline agents. US2006/0194955 shows that the mutated ligands can better withstandproteases thus reducing ligand leakage in the separation process.Another publication, US 2006/0194950 shows that the alkali stable SpAdomains can be further modified such that the ligands lacks affinity forFab but retains Fc affinity, for example by a G29A mutation.

Historically the native protein A containing 5 IgG binding domains wasused for production of all protein A affinity media. Using recomenbandtechnology a number of protein A construct have been produced allcontaining 4 or 5 IgG binding domains. A recent study showed thatdimeric ligands have a similar, or increased binding capacity comparedto tetrameric ligands (WO 2010/080065).

There is still a need in this field to obtain a separation matrixcontaining protein ligands having an increased binding capacity.

SUMMARY OF THE INVENTION

One object of the present invention is to provide protein ligandscapable of binding immunoglobulins, such as IgG, IgA and/or IgM,preferably via their Fc-fragments. These ligands carry a substitution ofthe C-terminal most proline residue, in at least one of the monomericdomains of protein A (E, D, A, C, C) or protein Z, after the thirdalpha-helix, thus have a higher binding capacity, as compared to ligandswithout the substitution. Preferably, the ligands are multimeric, i.e.,containing more than one monomeric domains selected from protein A (E,D, A, C, C) and protein Z.

Another object of the invention is to provide an affinity separationmatrix, which comprises the ligands capable of binding immunoglobulins,such as IgG, IgA and/or IgM, preferably via their Fc-fragments, asdescribed above.

A further object of the invention is to provide a method for separatingone or more immunoglobulin containing proteins, using the currentaffinity matrix. By using affinity ligands with the substitution, themethod unexpectedly achieves increased binding capacity for the targetmolecules.

Thus the invention provides a method for either producing a purifiedproduct, such as a pure immunoglobulin fraction or alternatively aliquid from which the immunoglobulin has been removed, or to detect thepresence of immunoglobulin in a sample. The ligands according to theinvention exhibit an increased capacity, which renders the ligandsattractive candidates for cost-effective large-scale operation.

One or more of the above-defined objects can be achieved as described inthe appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of protein Z, with the proline atposition 57 shown in bold (SEQ ID NO: 2).

FIG. 2 a shows a hypothetical tetrameric ligand structure, each monomeris a domain from protein A (E, D, A, B, C) or protein Z, which due tothe presence of the C-terminal most proline in each monomer, contains a60° bend between each domain.

FIG. 2 b shows hypothetical binding between the ligands and IgG where asingle IgG binds to two monomeric domains (both Fc-chains represented bya circle, each binds to one monomeric domain).

FIG. 2 c shows a hypothetical, more linear and flexible multimericligand structure among the monomer domains due to the substitution ofthe C-terminal most proline in each domain with another amino acidresidue.

FIG. 2 d shows an alignment of the amino acid sequences from each of thefive domains of protein A.

FIG. 3 shows a representative chromatogram result for dynamic bindingcapacity assay.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “protein” is used herein to describe proteins as well asfragments thereof. Thus, any chain of amino acids that exhibits a threedimensional structure is included in the term “protein”, and proteinfragments are accordingly embraced.

The term “functional variant” of a protein means herein a variantprotein, wherein the function, in relation to the invention defined asaffinity and stability, are essentially retained. Thus, one or moreamino acids those are not relevant for said function may have beenexchanged.

The term “parental molecule” is used herein for the correspondingprotein in the form before a mutation according to the invention hasbeen introduced.

The term “structural stability” refers to the integrity ofthree-dimensional form of a molecule, while “chemical stability” refersto the ability to withstand chemical degradation.

The term “Fc fragment-binding” protein means that the protein is capableof binding to the Fc fragment of an immunoglobulin. However, it is notexcluded that an Fc fragment-binding protein also can bind otherregions, such as Fab regions of immunoglobulins.

In the present specification, if not referred to by their full names,amino acids are denoted with the conventional one-letter symbols.

Mutations are defined herein by the number of the position exchanged,preceded by the wild type or non-mutated amino acid and followed by themutated amino acid. Thus, for example, the mutation of an asparagine inposition 23 to a threonine is denoted N23T.

The present invention in one aspect relates to a method of separatingone or more immunoglobulin containing proteins from a liquid, whichmethod comprises (a) contacting the liquid with a separation matrixcomprising ligands immobilised to a support; (b) allowing theimmunoglobulin containing proteins to adsorb to the matrix byinteraction with the ligands; (c) an optional step of washing theadsorbed immunoglobulin containing proteins; and (d) recovering theimmunoglobulin containing proteins by contacting the matrix with aneluent which releases the proteins. The method provides increasedbinding capacity of the ligands to the immunoglobulin molecules by usinga ligand, each of which comprises one or more domains (i.e., monomers)of staphylococcal Protein A (SpA) (E, D, A, B, C) or protein Z or afunctional variant thereof, wherein the C-terminal most proline in atleast one of the one or more domains have been substituted with anyother amino acid.

The immunoglobulin-binding protein (i.e., ligand) can be any proteinwith a native immunoglobulin-binding capability, such as Staphylococcalprotein A (SpA) or Streptococcal protein G (SpG), or recombinantproteins containing IgG-binding domains of these proteins. For a reviewof other such proteins, see e.g. Kronvall, G., Jonsson, K. Receptins: anovel term for an expanding spectrum of natural and engineered microbialproteins with binding properties for mammalian proteins, J. Mol.Recognit. 1999 January-February; 12(1):38-44. The ligands can compriseone of more of the E, D, A, B and C domains of SpA. More preferably theligands comprise domain B of protein A or the engineered protein Z.

Every protein Z or protein A domain contains a proline close to theC-terminal (P57) (see e.g., FIG. 1, FIG. 2 d, and SEQ ID NO. 1-8).Prolines are known to give turns in proteins due to their structure,i.e. the side-chain is linked to the alpha nitrogen. This link restrictsto bonding angles of phi=˜60°, thus giving a turn in the protein. Aclassical example is seen in the hinge of IgG, where prolines are found.Thus, when proline is present at the C-terminal, every domain of proteinA or protein Z ends with a 60° angle in the bond, giving a turn betweendomains (FIG. 2 a-2 c)). The C-terminal most proline is neither involvedin Fc-binding (Graille et al, PNAS 2000, 97 (10): 5399-5404;Deisenhofer, Biochemistry 1981, 20 (9): 2361-2370) nor part of an alphahelix (impossible for a proline).

Thus the protein A/protein Z ligand's poor usage of the monomericdomains (one out of two are used at total overloading) could be due tothe structural limitation caused by the presence of the C-terminalproline in each domain, i.e., steric hindrance. There is a possibilitythat two domains in a ligand bind one IgG molecule, therefore blockingIgG access to every other domain (FIG. 2 b). Such binding could alsoimply a stronger binding of IgG to the ligand. Certain embodiments ofthe invention substitute the C-terminal most proline (P57 of SEQ ID NO:1 or 2) in each domain to achieve a higher usage of each domain, andthus an increased capacity of the ligand, possibly due to a more linearand/or flexible multimeric ligand structure among the monomeric domains(FIG. 2 c).

As shown in FIG. 2 d, the sequences among the five domains of Protein Aare highly related. There are no deletions or insertion, and many of thesubstitutions are conservative changes with minimal potential change tothe structure or function of the protein. For example, there are onlyfour changes between the B domain and the C domain, over the entire 58amino acid polypeptide. Thus, the C-terminal proline in each of thesedomains have a similar structural/functional contribution to protein Aor ligand containing the domain. Similarly, changes of the proline causea similar effect to the structure/function of the ligand containing sucha change.

In certain embodiments, the proline is substituted with an amino acidthat preferentially forms beta-sheets. Preferably, the proline issubstituted for a bulky amino acid or amino acid giving “stiffness”,e.g. Y, W, F, M, I, V, T. More preferably, the substitution is P57I.

In other embodiments, the proline is substituted with an amino acid thatis prone to formation of alpha helices, e.g. E, A, L, H, M, Q, K.

In certain preferred embodiments, the parental molecule comprises thesequence defined by SEQ ID NO: 1-8, or any functional variance thereof.

In certain embodiments, the C-terminal most proline residue in at leastone of the monomeric domains in a multimeric ligand is substituted. Inother embodiments, the C-terminal most proline residue in all themonomeric domains in a multimeric ligand is substituted.

In one embodiment, the ligands are also rendered alkali-stable, such asby mutating at least one asparagine residue of at least one of themonomeric domains of the SpA domain B or protein Z to an amino acidother than glutamine. As discussed earlier, US patent applicationpublication US 2005/0143566 discloses that when at least one asparagineresidue is mutated to an amino acid other than glutamine or asparticacid, the mutation confers the ligand an increased chemical stability athigh pH (e.g., N23T). Further, affinity media including these ligandscan better withstand cleaning procedures using alkaline agents. US2006/0194955 shows that the mutated ligands can also better withstandproteases thus reducing ligand leakage in the separation process. Thedisclosures of these applications are hereby incorporated by referencein their entirety.

In another embodiment, the ligand(s) so prepared lack any substantialaffinity for the Fab part of an antibody, while having affinity for theFc part. Thus, in certain embodiments, at least one glycine of theligands has been replaced by an alanine. US 2006/0194950 shows that thealkali stable domains can be further modified such that the ligandslacks affinity for Fab but retains Fc affinity, for example by a G29Amutation. The disclosure of the application is hereby incorporated byreference in its entirety. The numbering used herein of the amino acidsis the conventionally used in this field, exemplified by the position ondomain B of protein A, and the skilled person in this field can easilyrecognize the position to be mutated for each domain of E, D, A, B, C,or protein Z.

In an advantageous embodiment, the ligand is made of multimer copies ofdomain B, and the alkali-stability of domain B has been achieved bymutating at least one asparagine residue to an amino acid other thanglutamine (e.g. N23T); and contains a mutation of the amino acid residueat position 29 of the alkali-stable domain B, such as a G29A mutation.

In another embodiment, the ligand is made of multimer copies of proteinZ in which the alkali-stability has been achieved by mutating at leastone asparagine residue to an amino acid other than glutamine. In anadvantageous embodiment, the alkali-stability has been achieved bymutating at least the asparagine residue at position 23 to an amino acidother than glutamine.

As the skilled person in this field will easily understand, thesubstitution of P57, the mutations to provide alkaline-stability, andthe G to A mutation may be carried out in any order of sequence usingconventional molecular biology techniques. Further, the ligands can beexpressed by a vector containing a nucleic acid sequence encoding themutated protein ligands. Alternatively, they can also be made by proteinsynthesis techniques. Methods for synthesizing peptides and proteins ofpredetermined sequences are well known and commonly available in thisfield.

Thus, in the present invention, the term “alkali-stable domain B ofStaphylococcal Protein A” means an alkali-stabilized protein based onDomain B of SpA, such as the mutant protein described in US patentapplication publications US 2005/0143566 and US 2006/0194950; as well asother alkali-stable proteins of other origin but having a functionallyequivalent amino acid sequence.

As the skilled person will understand, the expressed protein should bepurified to an appropriate extent before being immobilized to a support.Such purification methods are well known in the field, and theimmobilization of protein-based ligands to supports is easily carriedout using standard methods. Suitable methods and supports will bediscussed below in more detail.

Accordingly, in one embodiment, a mutated protein according to theinvention comprises at least about 75%, such as at least about 80% orpreferably at least about 95%, of the sequence as defined in SEQ ID NOs:1 or 2, with the proviso that the asparagine mutation is not in position21.

In the present specification, SEQ ID NO: 1 defines the amino acidsequence of the B-domain of SpA:

Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn AlaPhe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn GluGlu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys AspAsp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu AlaLys Lys Leu Asn Asp Ala Gln Ala Pro Lys.SEQ ID NO: 2 defines a protein known as protein Z:

Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn AlaPhe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn GluGlu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys AspAsp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu AlaLys Lys Leu Asn Asp Ala Gln Ala Pro Lys.

Protein Z is a synthetic construct derived from the B-domain of SpA,wherein the glycine in position 29 has been exchanged for alanine, seee.g. Ståhl et al, 1999: Affinity fusions in biotechnology: focus onprotein A and protein G, in The Encyclopedia of Bioprocess Technology:Fermentation, Biocatalysis and Bioseparation. M. C. Fleckinger and S. W.Drew, editors. John Wiley and Sons Inc., New York, 8-22.

In one embodiment, the above described mutant protein is comprised ofthe amino acid sequence defined in SEQ ID NOs: 1 or 2, or is afunctional variant thereof, with a substitution at P57. In anotherembodiment, the above described mutant protein is comprised of the aminoacid sequence defined in SEQ ID NOs: 4-8, or is a functional variantthereof, with a substitution at P57. The term “functional variant” asused in this context includes any similar sequence, which comprises oneor more further variations in amino acid positions that have noinfluence on the mutant protein's affinity to immunoglobulins or itsimproved chemical stability in environments of increased pH-values.

In an advantageous embodiment, the present substitutions of P57 areselected from the group that consists of a bulky amino acid or aminoacid giving “stiffness”, e.g. Y, W, F, M, I, V, T; and wherein theparental molecule comprises the sequence defined by SEQ ID NO: 1-8, orany functional variance thereof. In other embodiments, P57 issubstituted with an amino acids that is prone to formation of alphahelices, e.g. E, A, L, H, M, Q, K, and wherein the parental moleculecomprises the sequence defined by SEQ ID NO: 1-8, or any functionalvariance thereof. More preferably, the substitution is P57I. Asmentioned above, in order to achieve a mutant protein useful as a ligandwith high binding capacity for a prolonged period of time in alkalineconditions, mutation of the asparagine residue in position 21 isavoided. In one embodiment, the asparagine residue in position 3 is notmutated.

In certain embodiments, the C-terminal most proline residue in at leastone of the monomers in a multimeric ligand are substituted. In otherembodiments, the C-terminal most proline residue in all the monomers ina multimeric ligand are substituted.

In one advantageous embodiment, an asparagine residue located between aleucine residue and a glutamine residue has also been mutated, forexample to a threonine residue. Thus, in one embodiment, the asparagineresidue in position 23 of the sequence defined in SEQ ID NO: 2 has beenmutated, for example to a threonine residue. In a specific embodiment,the asparagine residue in position 43 of the sequence defined in SEQ IDNO: 2 has also been mutated, for example to a glutamic acid. In theembodiments where amino acid number 43 has been mutated, it appears tomost advantageously be combined with at least one further mutation, suchas N23T.

Thus, the invention encompasses the above-discussed monomeric mutantproteins. However, such protein monomers can be combined into multimericligands, such as dimers, trimers, tetramers, pentamers, hexamers etc.Accordingly, another aspect of the present invention is a multimercomprised of at least one of the mutated proteins according to theinvention together with one or more further units, preferably alsomutant proteins according to the invention. Thus, the present inventionis e.g. a dimer comprised of two repetitive units, or a tetramercomprised of four repetitive units.

In certain embodiments, the multimeric ligands contain two or more, suchas 2-4, copies of the same monomeric domain from domain E, D, A, B, C ofprotein A, or protein Z, or any functional variants.

In other embodiments, the multimeric ligands contain two or more, suchas 2-4, different monomeric domains selected from domain E, D, A, B, Cof protein A, or protein Z, or any functional variants.

In one embodiment, the multimer according to the invention comprisesmonomer units linked by a stretch of amino acids preferably ranging from0 to 15 amino acids, such as 0-10 or 5-10 amino acids. The nature ofsuch a link should preferably not destabilize the spatial conformationof the protein units. Furthermore, said link should preferably also besufficiently stable in alkaline environments not to impair theproperties of the mutated protein units.

In another embodiment, the present dimeric ligands comprise the sequenceof SEQ ID NO: 3:

AlaGlnValAspAlaLysPheAspLysGluGlnGlnAsnAlaPheTyrGluIleLeuHisLeuProAsnLeuThrGluGluGlnArgAsnAlaPheIleGlnSerLeuLysAspAspProSerGlnSerAlaAsnLeuLeuAlaGluAlaLysLysLeuAsnAspAlaGlnAlaIleLysValAspAlaLysPheAspLysGluGlnGlnAsnAlaPheTyrGluIleLeuHisLeuProAsnLeuThrGluGluGlnArgAsnAlaPheIleGlnSerLeuLysAspAspProSerGlnSerAlaAsnLeuLeuAlaGluAlaLysLysLeuAsn AspAlaGlnAlaIleLysCysIn a further embodiment, the dimeric ligands comprise the sequence ofSEQ ID NO: 9:

AlaGlnValAspAsnLysPheAsnLysGluGlnGlnAsnAlaPheTyrGluIleLeuHisLeuProAsnLeuThrGluGluGlnArgAsnGlyPheIleGlnSerLeuLysAspAspProSerValSerLysGluIleLeuAlaGluAlaLysLysLeuAsnAspAlaGlnAlaIleLysValAspAsnLysPheAsnLysGluGlnGlnAsnAlaPheTyrGluIleLeuHisLeuProAsnLeuThrGluGluGlnArgAsnGlyPheIleGlnSerLeuLysAspAspProSerValSerLysGluIleLeuAlaGluAlaLysLysLeuAsn AspAlaGlnAlaIleLysCysIn another embodiment, the tetrameric ligands comprise the sequence ofSEQ ID NO: 10:

AlaGlnValAspAlaLysPheAspLysGluGlnGlnAsnAlaPheTyrGluIleLeuHisLeuProAsnLeuThrGluGluGlnArgAsnAlaPheIleGlnSerLeuLysAspAspProSerGlnSerAlaAsnLeuLeuAlaGluAlaLysLysLeuAsnAspAlaGlnAlaIleLysValAspAlaLysPheAspLysGluGlnGlnAsnAlaPheTyrGluIleLeuHisLeuProAsnLeuThrGluGluGlnArgAsnAlaPheIleGlnSerLeuLysAspAspProSerGlnSerAlaAsnLeuLeuAlaGluAlaLysLysLeuAsnAspAlaGlnAlaIleLysValAspAlaLysPheAspLysGluGlnGlnAsnAlaPheTyrGluIleLeuHisLeuProAsnLeuThrGluGluGlnArgAsnAlaPheIleGlnSerLeuLysAspAspProSerGlnSerAlaAsnLeuLeuAlaGluAlaLysLysLeuAsnAspAlaGlnAlaIleLysValAspAlaLysPheAspLysGluGlnGlnAsnAlaPheTyrGluIleLeuHisLeuProAsnLeuThrGluGluGlnArgAsnAlaPheIleGlnSerLeuLysAspAspProSerGlnSerAlaAsnLeuLeuAlaGluAlaLysLysLeuAsnAspAlaGlnAlaIleLysCys

The current invention unexpectedly found that when comparing thecapacity of the ligands, a substitution of the C-terminal proline (P57)in at least one of the monomeric domains of the ligand provides a highercapacity compared to non-substituted ligands.

One aspect of the invention relates to a ligand for separation ofimmunoglobulin containing proteins. The ligand comprises one or moredomains of staphylococcal protein A (SpA) (E, D, A, C, C) or protein Zor a functional variant thereof, wherein the C-terminal most proline inat least one of the domains has been substituted with another aminoacid. Such ligands provide for an increased immunoglobulin capacity whenimmobilised on solid support materials, compared to ligands which do nothave this substitution. The ligand can be constructed according to anyembodiment described above.

In one aspect, the invention relates to a matrix for affinityseparation, which matrix comprises ligands that compriseimmunoglobulin-binding protein coupled to a solid support. Preferably,the C-terminal most proline residue, in at least one of the monomericdomains of protein A (E, D, A, C, C) or protein Z, after the thirdalpha-helix, P57 residue has been substituted to another amino acid.Preferably, the ligands are multimeric, i.e., containing more than onemonomeric domains selected from protein A (E, D, A, C, C) and protein Z.The present matrix, when compared to a matrix without the substitution,exhibits an increased binding capacity. The mutated protein ligand ispreferably an Fc-fragment-binding protein, and can be used for selectivebinding of IgG, IgA and/or IgM, preferably IgG.

The matrix according to the invention can comprise the mutant protein asdescribed above in any embodiment thereof as ligand. In the mostpreferred embodiment, the ligands present on the solid support comprisea substitution as described above.

The solid support of the matrix according to the invention can be of anysuitable well-known kind. A conventional affinity separation matrix isoften of organic nature and based on polymers that expose a hydrophilicsurface to the aqueous media used, i.e. expose hydroxy (—OH), carboxy(—COOH), carboxamido (—CONH₂, possibly in N-substituted forms), amino(—NH₂, possibly in substituted form), oligo- or polyethylenoxy groups ontheir external and, if present, also on internal surfaces. In oneembodiment, the polymers may, for instance, be based on polysaccharides,such as dextran, starch, cellulose, pullulan, agar, agarose etc, whichadvantageously have been cross-linked, for instance with bisepoxides,epihalohydrins, 1,2,3-trihalo substituted lower hydrocarbons oraccording to the methods described in U.S. Pat. No. 6,602,990, toprovide a suitable porosity and rigidity. In the most preferredembodiment, the solid support is porous agar or agarose beads. Thesupports used in the present invention can easily be prepared accordingto standard methods, such as inverse suspension gelation (S Hjertén:Biochim Biophys Acta 79(2), 393-398 (1964). Alternatively, the basematrices are commercially available products, such as Sepharose™ FF orSepharose HP (GE Healthcare). In an embodiment, which is especiallyadvantageous for large-scale separations, the support has been adaptedto increase its rigidity, and hence renders the matrix more suitable forhigh flow rates.

Alternatively, the solid support is based on synthetic polymers, such aspolyvinyl alcohol, polyhydroxyalkyl acrylates, polyhydroxyalkylmethacrylates, polyacrylamides, polymethacrylamides etc. In case ofhydrophobic polymers, such as matrices based on divinyl andmonovinyl-substituted benzenes, the surface of the matrix is oftenhydrophilised to expose hydrophilic groups as defined above to asurrounding aqueous liquid. Such polymers are easily produced accordingto standard methods, see e.g. “Styrene based polymer supports developedby suspension polymerization” (R Arshady: Chimica e L'Industria 70(9),70-75 (1988)). Alternatively, a commercially available product, such asSOURCE™ (GE Healthcare) is used.

In another alternative, the solid support according to the inventioncomprises a support of inorganic nature, e.g. silica, zirconium oxideetc.

In yet another embodiment, the solid support is in another form such asa surface, a chip, capillaries, or a filter.

As regards the shape of the matrix according to the invention, in oneembodiment the matrix is in the form of a porous monolith. In analternative embodiment, the matrix is in beaded or particle form thatcan be porous or non-porous. Matrices in beaded or particle form can beused as a packed bed or in a suspended form. Suspended forms includethose known as expanded beds and pure suspensions, in which theparticles or beads are free to move. In case of monoliths, packed bedand expanded beds, the separation procedure commonly followsconventional chromatography with a concentration gradient. In case ofpure suspension, batch-wise mode will be used.

The ligand may be attached to the support via conventional couplingtechniques utilising, e.g. amino and/or carboxy groups present in theligand. Bisepoxides, epichlorohydrin, CNBr, N-hydroxysuccinimide (NHS)etc are well-known coupling reagents. Between the support and theligand, a molecule known as a spacer can be introduced, which willimprove the availability of the ligand and facilitate the chemicalcoupling of the ligand to the support. Alternatively, the ligand may beattached to the support by non-covalent bonding, such as physicaladsorption or biospecific adsorption. The ligand content of the matrixmay be 5-15 mg/ml matrix and can advantageously be 5-10 mg/ml.

In an advantageous embodiment, the present ligand has been coupled tothe support by thioether bonds. Methods for performing such coupling arewell-known in this field and easily performed by the skilled person inthis field using standard techniques and equipment. In an advantageousembodiment, the ligand is firstly provided with a terminal cysteineresidue for subsequent use in the coupling. The skilled person in thisfield also easily performs appropriate steps of purification.

In certain embodiments of the invention, the conditions for theadsorption step may be any conventionally used, appropriately adapteddepending on the properties of the target antibody such as the pIthereof. The optional wash step can be performed using a buffer commonlyused such as a PBS buffer.

The elution may be performed by using any commonly used buffer.

The present method is useful to capture target antibodies, such as afirst step in a purification protocol of antibodies which are e.g. fortherapeutic or diagnostic use. In one embodiment, at least 75% of theantibodies are recovered. In an advantageous embodiment, at least 80%,such as at least 90%, and preferably at least 95% of the antibodies arerecovered using an eluent having a suitable pH for the particular ligandsystem. The present method may be followed by one or more additionalsteps, such as other chromatography steps. Thus, in a specificembodiment, more than about 98% of the antibodies are recovered.

As discussed earlier, for either SpA (domain E, D, A, B, C) or protein Zligand, when at least one asparagine residue is mutated to an amino acidother than glutamine or aspartic acid, affinity media including thesemutant ligands can better withstand cleaning procedures using alkalineagents (US 2005/0143566). The increased stability means that the mutatedprotein's initial affinity for immunoglobulin is essentially retainedfor a prolonged period of time. Thus its binding capacity will decreasemore slowly than that of the parental molecule in an alkalineenvironment. The environment can be defined as alkaline, meaning of anincreased pH-value, for example above about 10, such as up to about 13or 14, i.e. from 10-13 or 10-14, in general denoted alkaline conditions.Alternatively, the conditions can be defined by the concentration ofNaOH, which can be up to about 1.0 M, such as 0.7 M or specificallyabout 0.5 M, accordingly within a range of 0.7-1.0 M.

Thus, the affinity to immunoglobulin i.e. the binding properties of thepresent ligand, in the presence of the asparagine mutation as discussed,and hence the capacity of the matrix, is not essentially changed in timeby treatment with an alkaline agent. Conventionally, for a cleaning inplace treatment of an affinity separation matrix, the alkaline agentused is NaOH and the concentration thereof is up to 0.75 M, such as 0.5M. Thus, its binding capacity will decrease to less than about 70%,preferably less than about 50% and more preferably less than about 30%,such as about 28%, after treatment with 0.5 M NaOH for 7.5 h.

In a further aspect, the present invention relates to a method ofisolating an immunoglobulin, such as IgG, IgA and/or IgM, wherein aligand or a matrix according to the invention is used. Thus, theinvention encompasses a process of chromatography, wherein at least onetarget compound is separated from a liquid by adsorption to a ligand ormatrix described above. The desired product can be the separatedcompound or the liquid. Thus, this aspect of the invention relates toaffinity chromatography, which is a widely used and well-knownseparation technique. In brief, in a first step, a solution comprisingthe target compounds, preferably antibodies as mentioned above, ispassed over a separation matrix under conditions allowing adsorption ofthe target compound to ligands present on said matrix. Such conditionsare controlled e.g. by pH and/or salt concentration i.e. ionic strengthin the solution. Care should be taken not to exceed the capacity of thematrix, i.e. the flow should be sufficiently slow to allow asatisfactory adsorption. In this step, other components of the solutionwill pass through in principle unimpeded. Optionally, the matrix is thenwashed, e.g. with an aqueous solution, in order to remove retainedand/or loosely bound substances. The present matrix is mostadvantageously used with an intermediate washing step utilizingadditives such as solvents, salts or detergents or mixture there of. Ina next step, a second solution denoted an eluent is passed over thematrix under conditions that provide desorption i.e. release of thetarget compound. Such conditions are commonly provided by a change ofthe pH, the salt concentration i.e. ionic strength, hydrophobicity etc.Various elution schemes are known, such as gradient elution andstep-wise elution. Elution can also be provided by a second solutioncomprising a competitive substance, which will replace the desiredantibody on the matrix. For a general review of the principles ofaffinity chromatography, see e.g. Wilchek, M., and Chaiken, I. 2000. Anoverview of affinity chromatography. Methods Mol. Biol. 147: 1-6.

EXAMPLES

Below, the present invention will be described by way of examples, whichare provided for illustrative purposes only and accordingly are not tobe construed as limiting the scope of the present invention as definedby the appended claims. All references given below and elsewhere in thisapplication are hereby included herein by reference.

Example 1

Prototypes

Mutant Z(P57I)2: ligand dimers containing two copies of protein Z, eachcontaining the P57I substitution (Z(P57I)2) (SEQ ID No. 3), with liganddensity of 5.8 mg/ml.

Mutant Z(P57I)4: ligand tetramers containing four copies of protein Z,each containing the P57I substitution (Z(P57I)4) (SEQ ID No. 10), withligand density of 9.7 mg/ml.

Mutant C(P57I)2: ligand dimers containing two copies of the Protein A Cdomain, each containing the P57I substitution (C(P57I)2) (SEQ ID No. 9),with ligand density of 7.2 mg/ml.

Regular Z2: ligand dimers containing two copies of protein Z (Z2 reg)(SEQ ID No. 3 but without the P57I substitutions), with ligand densityof 6.1 mg/ml.

2 ml of each resin was packed in Tricorn 5 100 columns

Protein

Gammanorm 165 mg/ml (Octapharma), diluted to 1 mg/ml in Equilibrationbuffer.

Equilibration Buffer

APB Phosphate buffer 20 mM+0.15M NaCl, pH 7,4 (Elsichrom AB)

Adsorption Buffer

APB Phosphate buffer 20 mM+0.15M NaCl, pH 7.4 (Elsichrom AB).

Elution Buffer

APB Citrate buffer 0.1M, pH 3 (Elsichrom AB).

CIP

0.1M NaOH.

Experimental Details and Results:

Mutagenesis of Protein

Site-directed mutagenesis was performed by PCR using an oligonucleotidecoding for the pro-line replacement. As template a plasmid containing asingle domain of either Z or C was used. The PCR fragments were ligatedinto an E. coli expression vector (pGO). DNA sequencing was used toverify the correct sequence of inserted fragments.

To form multimers of Z(P57I) and C(P57I) an Acc I site located in thestarting codons (GTA GAC) of the C or Z domain was used, correspondingto amino acids VD. pGO Z(P57I)1 and pGO C(P57I)1 were digested with AccI and CIP treated. Acc I sticky-ends primers were designed, specific foreach variant, and two overlapping PCR products were generated from eachtemplate. The PCR products were purified and the concentration wasestimated by comparing the PCR products on a 2% agarose gel. Equalamounts of the pair wise PCR products were hybridized (90° C.->25° C. in45 min) in ligation buffer. The resulting product consists approximatelyto ¼ of fragments likely to be ligated into an Acc I site (correct PCRfragments and/or the digested vector). After ligation and transformationcolonies were PCR screened to identify constructs containing Z(P57I)2,Z(P57I)4, C(P57I)2 and C(P57I)4. Positive clones were verified by DNAsequencing.

Construct Expression and Purification

The constructs were expressed in the bacterial periplasm by fermentationof E. coli K12 in standard media. After fermentation the cells wereheat-treated to release the periplasm content into the media. Theconstructs released into the medium were recovered by microfiltrationwith a membrane having a 0.2 μm pore size.

Each construct, now in the permeate from the filtration step, waspurified by affinity. The permeate was loaded onto a chromatographymedium containing immobilized IgG. The loaded product was washed withphosphate buffered saline and eluted by lowering the pH.

The elution pool was adjusted to a neutral pH and reduced by addition ofdithio threitol. The sample was then loaded onto an anion exchanger.After a wash step the construct was eluted in a NaCl gradient toseparate it from any contaminants. The elution pool was concentrated byultra-filtration to 40-50 mg/ml.

The purified ligands were analyzed with LC-MS to determine the purityand to ascertain that the molecular weight corresponded to the expected(based on the amino acid sequence).

Activation

The base matrix used was rigid crosslinked agarose beads of 85 micronsaverage diameter, prepared according to the methods of U.S. Pat. No.6,602,990 and with a pore size corresponding to an inverse gelfiltration chromatography Kav value of 0.70 for dextran of Mw 110 kDa,according to the methods described in Gel Filtration Principles andMethods, Pharmacia LKB Biotechnology 1991, pp 6-13.

25 mL (g) of drained base matrix, 10.0 mL distilled water and 2.02 gNaOH (s) was mixed in a 100 mL flask with mechanical stirring for 10 minat 25° C. 4.0 mL of epichlorohydrin was added and the reactionprogressed for 2 hours. The activated gel was washed with 10 gelsediment volumes (GV) of water.

Coupling

To 20 mL of ligand solution (50 mg/mL) in a 50 ml Falcon tube, 169 mgNaHCO₃, 21 mg Na₂CO₃, 175 mg NaCl and 7 mg EDTA, was added. The Falcontube was placed on a roller table for 5-10 min, and then 77 mg of DTEwas added. Reduction proceeded for >45 min The ligand solution was thendesalted on a PD10 column packed with Sephadex G-25. The ligand contentin the desalted solution was determined by measuring the 276 nm UVabsorption.

The activated gel was washed with 3-5 GV {0.1 M phosphate/1 mM EDTA pH8.6} and the ligand was then coupled according to the method describedin U.S. Pat. No. 6,399,750. All buffers used in the experiments had beendegassed by nitrogen gas for at least 5-10 min.

After immobilisation the gels were washed 3×GV with distilled water. Thegels+1 GV {0.1 M phosphate/1 mM EDTA/10% thioglycerol pH 8.6} was mixedand the tubes were left in a shaking table at room temperature overnight. The gels were then washed alternately with 3×GV {0.1 M TRIS/0.15M NaCl pH 8.6} and 0.5 M HAc and then 8-10×GV with distilled water. Gelsamples were sent to an external laboratory for amino acid analysis andthe ligand content (mg/ml gel) was calculated from the total amino acidcontent.

Capacity Determination

The breakthrough capacity was determined with an ÄKTAExplorer 10 systemat a residence time of 2.4 and 6 min as standard. Adsorption buffer wasrun through the bypass column until a stable baseline was obtained. Thiswas done prior to auto zeroing. Sample was applied to the column until a100% UV signal was obtained. Then, adsorption buffer was applied againuntil a stable baseline.

The column was equilibrated with adsorption buffer prior to loading withsample until a UV signal of 85% of maximum absorbance was reached. Thecolumn was then washed with adsorption buffer until a UV signal of 20%of maximum absorbance at flow rate 0.5 ml/min. The protein was elutedwith 10 CV elution buffer at flow rate 0.5 ml/min. Then the column wascleaned with 0.1M NaOH at flow rate 0.5 ml/min and re-equilibrated withadsorption buffer prior to cleaning with 20% ethanol. The last step wasto check the sample concentration by loading sample through the bypasscolumn until a 100% UV signal was obtained.

For calculation of breakthrough capacity at 10%, the equation below wasused. That is the amount of IgG that is loaded onto the column until theconcentration of IgG in the column effluent is 10% of the IgGconcentration in the feed.

$q_{10\%} = {\frac{C_{0}}{V_{c\;}}\left\lbrack {V_{app} - V_{sys} - {\int_{V_{sys}}^{V_{app}}{\frac{{A(V)} - A_{sub}}{A_{100\%} - A_{sub}}*{\mathbb{d}v}}}} \right\rbrack}$

A_(100%)=100% UV signal;

A_(sub)=absorbance contribution from non-binding IgG subclass;

A(V)=absorbance at a given applied volume;

V_(c)=column volume;

V_(app)=volume applied until 10% breakthrough;

V_(sys)=system dead volume;

C₀=feed concentration.

A typical run for dynamic binding capacity is shown in FIG. 3. Thedynamic binding capacity (DBC) at 10% breakthrough was calculated andthe appearance of the curve was studied. The curve was also studiedregarding binding, elution and CIP peak.

The dynamic binding capacity (DBC) was calculated for 5, 10 and 80%breakthrough. Results are shown in Table 1. Higher capacity was observedfor ligands with P57I substitution as compared to the parental P57ligand.

TABLE 1 Summary of results. Prototype Ligand (two columns density Qb5Qb10 Qb80 Res were packed (mg/ml (mg/ml (mg/ml (mg/ml time and tested)resin) resin) resin) resin) (min) Z(P57I)2#1 5.8 31.5 34.3 51 2.4Z(P57I)2#2 5.8 33.5 35.7 51.8 2.4 Z(P57I)2#2 5.8 32.4 34.6 50.3 2.4Z(P57I)2#2 5.8 34.0 36.2 53.4 2.4 Z(P57I)2#1 5.8 44.1 45.6 53.5 6.0Z(P57I)2#2 5.8 43.9 45.6 53.6 6.0 Z(P57I)2#2 5.8 42.6 44.6 na 6.0Z(P57I)2#2 5.8 45.4 46.6 54.5 6.0 Z2reg#1 6.1 28.5 31.4 43.8 2.4 Z2reg#26.1 29.9 32.2 46.1 2.4 Z2reg#1 6.1 38.1 40.6 46.7 6.0 Z2reg#2 6.1 38.540.4 47.1 6.0 C(P57I)2 7.2 36.9 40.2 63.7 2.4 C(P57I)2 7.2 52.6 55.266.9 6.0 Z(P57I)4 9.7 35.2 39.0 68.0 2.4 Z(P57I)4 9.7 54.7 57.1 72.4 6.0

The above examples illustrate specific aspects of the present inventionand are not intended to limit the scope thereof in any respect andshould not be so construed. Those skilled in the art having the benefitof the teachings of the present invention as set forth above, can effectnumerous modifications thereto. These modifications are to be construedas being encompassed within the scope of the present invention as setforth in the appended claims. It is further understood that features ofdifferent embodiments can be combined.

What is claimed is:
 1. A ligand for separation of immunoglobulincontaining proteins, which ligand comprises one or more monomericdomains of staphylococcal protein A (SpA) (E, D, A, B, C) or protein Z,wherein the proline nearest the C terminus in at least one of thedomains has been substituted with any other amino acid.
 2. The ligand ofclaim 1, wherein said one or more domains of protein A or protein Z aretwo or more copies from the same domain of SpA (E, D, A, B, C) orprotein Z respectively.
 3. The ligand of claim 1, wherein the prolinenearest the C terminus in at least one of the monomers in a multimericligand has been substituted with another amino acid.
 4. The ligand ofclaim 1, wherein the proline nearest the C terminus in all the monomersin a multimeric ligand have been substituted with another amino acid. 5.The ligand of claim 1, wherein the proline nearest the C terminus in atleast one monomer is substituted with an amino acid selected from Y, W,F, M, I, V, T.
 6. The ligand of claim 1, wherein the proline nearest theC terminus in at least one monomer is substituted with isoleucine. 7.The ligand of claim 1, wherein the proline nearest the C terminus in atleast one monomer is substituted with an amino acid selected from E, A,L, H, M, Q, K.
 8. The ligand of claim 1, wherein the ligand achievesalkali stability by mutating at least one asparagine residue to an aminoacid other than glutamine.
 9. The ligand of claim 1, wherein the ligandhas affinity for the Fc part of an immunoglobulin but lacks affinity forthe Fab part of an immunoglobulin.
 10. The ligand of claim 1, wherein atleast one glycine of said ligands has been replaced by an alanine. 11.The ligand of claim 1, wherein the ligand is protein Z in which thealkali-stability has been achieved by mutating at least one asparagineresidue to an amino acid other than glutamine.
 12. The ligand of claim11, wherein the alkali-stability of protein Z has been achieved bymutating at least the asparagine residue at position 23 to an amino acidother than glutamine.
 13. The ligand of claim 1, wherein said multimeris a dimer or a tetramer.